Studies of the Acetyl Coenzyme a Synthetase Reaction. I. Isolation and Characterization of Enzyme-bound Acetyl Adenylate.

The acyl adenylates participate in a variety of synthetase reactions, and their recent isolation from reactions containing substrate quantities of enzyme suggests that these compounds act as enzyme-bound intermediates (2-7). However, such evidence does not esclude the possibility, emphasized by Boyer, Mills, and Fromm, that the adenylate occurs only as a free side product that is in equilibrium with an enzyme-bound intermediate (8,9). For this reason an attempt was made with acetyl-CoA synthetase to isolate an enzyme-bound intermediate. This effort was facilitated greatly by the recent preparation of an acetyl-CoA synthetase in relatively homogeneous form (10). The present report presents strong evidence that an enzyme-bound intermediate participates in the acetyl-CoA synthetase reaction and that it is acetyl adenylate. Certain characteristics of its formation in partial Reactions 1 and 2 are also described.